A total of 115 Japanese NAFLD patients, 65 with NASH and 50 with simple steatosis, and 441 healthy control subjects were recruited to participate in this study at Yokohama City University Hospital. All control subjects were confirmed to have normal liver function, not to have viral hepatitis, and not to be alcoholics. The control subjects all had a BMI < 25 kg/m2, normal fasting glucose (<110 mg/dl), serum triglycerides (<150 mg/dl), and serum HDL cholesterol (>40 mg/dl) levels, and normal systolic (<130 mmHg) and diastolic blood pressure (<85 mmHg). Liver biopsy was performed in all 115 NAFLD patients, and the liver biopsy tissue obtained was stained with hematoxylin-eosin, reticulin stain, and Masson trichrome stain. The histological criterion used to make the diagnosis of NAFLD was the presence of macrovesicular fatty change in hepatocytes with displacement of the nucleus to the edge of the cell . When more than 5% of hepatocytes were affected by macrovesicular steatosis, the patient was diagnosed as having either steatosis or steatohepatitis. The criteria used to make the diagnosis of steatohepatitis were the presence of lobular inflammation and the presence of either ballooning cells or perisinusoidal/pericellular fibrosis in zone 3 of the hepatic acinus, in addition to steatosis [20, 21]. Patients with any of the following diseases were excluded from participation in this study: infectious hepatitis (chronic hepatitis C infection or concurrent active hepatitis B virus), autoimmune hepatitis, primary biliary cirrhosis (PBC), sclerosing cholangitis, hemochromatosis, α1-antitrypsin deficiency, Wilson's disease, drug-induced hepatitis, and alcoholic hepatitis, and heavy alcohol consumers (current or past daily consumption of more than 20 g alcohol per day). No patients had clinical evidence of hepatic decompensation, such as hepatic encephalopathy, ascites, variceal bleeding, or a serum billirubin level greater than twice the upper limit of normal.
Written informed consent was obtained from all subjects before their entry into this study. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Research Committee of Yokohama City Hospital.
Physical and laboratory evaluation
The body weight and height of the patients were measured with a calibrated scale after requesting them to remove their shoes and any heavy clothing. A venous blood sample was obtained from the patients after an overnight fast (12 hours) to measure their serum AST, ALT, glucose, immunoreactive insulin (IRI), hemoglobin A1c (HbA1c), total cholesterol, HDL cholesterol, and triglyceride levels. All laboratory biochemical parameters were measured with a conventional automated analyzer. Visceral fat area (VFA) and subcutaneous fat area (SFA) were measured by computed tomography (CT).
DNA preparation and SNP genotyping
Genomic DNA was prepared from each blood sample by using a commercial genomic DNA extraction kit (TALENT s.r.l., Trieste, Italy). The PPARGC1A SNPs were selected from the IMS-JST (Institute of Medical Science-Japan Science and Technology Agency) SNP database . We selected the 15 SNPs with a minor allele frequency greater than 0.2 and whose expected allele frequencies did not widely diverge from Hardy-Weinberg equilibrium (p > 0.001). Invader probes (Third Wave Technologies, Madison, WI) were synthesized for these SNPs, and the SNPs were genotyped in the cases and controls by a combination of multiplex PCR and the Invader assay, as described previously .
Real-time RT-PCR for measurement of PPARGCA1 mRNA expression
Total RNA was isolated from samples of liver biopsy specimens by using an RNeasy Mini Kit (Quiagen, Hilden, Germany) according to the manufacturer's instructions. The protocol included a DNase treatment step to remove genomic DNA. The RNA was assessed quantitatively by measuring relative absorbance at 260 nm and 280 nm, and qualitatively by ethidium bromide agarose-gel electrophoresis. Reverse transcription to produce cDNA was performed by using a TaqMan Reverse Transcription Reagents (Applied Biosystems, Foster City, CA, USA), according to the manufacturer's instructions. The reaction mixtures (100 μl) contained 2.5 μg of total RNA, and after allowing the reaction to proceed for 50 minutes at 48°C, the reverse transcriptase was inactivated by heating the samples to 95°C for 5 minutes.
Real-time quantitative RT-PCR was performed in triplicate by using an ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA, USA) and SYBR Green PCR Master Mix according to the manufacturer's protocol. The following primers were used: PPARGC1A (F, 5'-TCTGACGTGACCATGGTGTT-3'; R, 5'-CATTCCAGGGACTCCACACT-3'). The primers were designed with Primer Express software (Applied Biosystems, Foster City, CA, USA) based on the sequence data obtained from the GenBank database. β-actin (Applied Biosystems, Foster City, CA, USA) was used as a reference; i.e., each sample was normalized on the basis of its β-actin content. Thermal cycling was performed as follows: initial denaturation at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15seconds and 60°C for 1 minute.
For each case-control study, the frequencies of the genotypes or the alleles were compared between cases and controls in three different modes by means of the χ2 test. In the first mode (allele frequency mode), allele frequencies were compared between cases and controls by means of a 2 × 2 contingency table. In the second mode (recessive mode), the frequencies of the subjects who were homozygous for allele 1 were compared with the rest by means of a 2 × 2 contingency table, while in the third mode (dominant mode) the frequencies of the subjects who had allele 1 (allele 1 homozygotes and heterozygotes) were compared with the rest by means of a 2 × 2 contingency table. The odds ratio (OR) and its 95% confidence interval (CI) were calculated by Woolf's method. Conformity to the Hardy-Weinberg equilibrium was assessed by the χ2 test . Haplotype blocks were calculated using Haploview 3.2 software . Physical and laboratory data are reported as means ± standard deviation (SD).